Legesse et al. BMC Infectious Diseases 2010, 10:354
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RESEARCH ARTICLE
Open Access
Performance of QuantiFERON-TB Gold In-Tube
(QFTGIT) for the diagnosis of Mycobacterium
tuberculosis (Mtb) infection in Afar Pastoralists,
Ethiopia
Mengistu Legesse1,3*, Gobena Ameni1, Gezahegne Mamo2,3, Girmay Medhin1, Gunnar Bjune3, Fekadu Abebe3
Abstract
Background: Currently, T-cell based gamma interferon (IFNg) release assays (IGRAs) are acknowledged as the best
methods available for the screening of latent tuberculosis infection (LTBI) and also as aid for the diagnosis of active
tuberculosis (TB). To our information, the performance of these diagnostic tests has not been evaluated in Ethiopia.
Therefore, the intent of this study was to evaluate the performance of QuantiFERON-TB Gold In-Tube (QFTGIT) in
patients clinically suspected of active pulmonary TB (PTB) as well as in healthy subjects prior to its utilization for
the epidemiological study of active TB and LTBI in Afar pastoralists.
Methods: The sensitivity of QFTGIT was evaluated in 140 subjects who were clinically suspected of PTB using the
cut-off value recommended by the manufacturer (≥ 0.35 IU/ml) and disease-specific cut-off value. Sputum culture
result was used as a gold standard. The specificity of the test was evaluated both in patients and in 55 tuberculin
skin test (TST) negative healthy subjects.
Results: Out of the 140 study participants, 37 (26.4%) were positive for active PTB by culture. Out of the 37
subjects who had positive results by culture, 6 individuals were HIV-seropositive. Out of the 103 subjects who were
negative by culture, 6 subjects had indeterminate results and 21 were HIV-seropositive. The performance of the
test was assessed using data from 107 (31 culture positive and 76 culture negative) individuals who were clinically
suspected of PTB and HIV-seronegatives. Using the manufacturer recommended cut-off value, the sensitivity of the
test was 64.5% (20/31), while its specificity was 36.8% (28/76). The sensitivity of the test was increased to 77.4%,
while the specificity was reduced to 23.7% using a cut-off value ≥ 0.1 IU/ml of IFNg as disease-specific cut-off
value. In TST negative healthy subjects, the specificity of the test was 58.2%.
Conclusion: Our findings revealed a low sensitivity of QFTGIT in the diagnosis of Mycobacterium tuberculosis (Mtb)
infection in the present study area using the cut-off value recommended by the manufacturer. Nevertheless, the
sensitivity increased from 64.5% to 77.4% by lowering the cut-off value recommended by the manufacturer to
≥ 0.1 IU/ml of IFNg level. Hence, it is of practical importance to evaluate the performance of QFTGIT in population
under different settings prior to its application either for the diagnosis of active TB or LTBI.
Background
Approximately one third of the world’s population is
harbouring latent tuberculosis infection (LTBI) though
most of the infected individuals do not develop active
* Correspondence: dlegessem@yahoo.com
1
Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis
Ababa, Ethiopia
Full list of author information is available at the end of the article
form of the disease [1]. Factors like HIV/AIDS and
undernutrition that can affect the host immune response
are substantially contributing to the re-activation of
LTBI [2,3]. In the era of HIV/AIDS pandemic, there is a
fear that over 200 million active tuberculosis (TB) cases
and 35 million deaths might occur globally by 2020, if
TB control is not increased [4]. Hence, screening and
treating individuals harbouring LTBI may minimize the
© 2010 Legesse et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Legesse et al. BMC Infectious Diseases 2010, 10:354
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risk of subsequent development to active TB, especially
in individuals at high risk of progression though this
strategy is less applicable in areas where the disease is
endemic, because of factors like high prevalence of
LTBI, HIV/AIDS, malnutrition as well as the high cost
of IGRAs.
Tuberculin skin test (TST), which has been used for
the diagnosis of LTBI for a century [5], is relatively
inexpensive and does not require laboratory facilities to
perform. Nevertheless, its limitations like high rate of
false-positive results in bacille Calmette-Guérin (BCG)
vaccinated or in atypical environmental mycobacterium
infected individuals as well as high rate of false-negative
results among immunosuppressed individuals made difficult its utilization for the screening of LTBI, especially
in regions where BCG vaccination and HIV/AIDS are
common [6,7].
Alternative diagnostic methods to the TST, T-cell
based gamma interferon (IFNg) release assays (IGRAs)
have been developed and approved mainly for the diagnosis of LTBI and also as aid for the diagnosis of active
TB [8]. Currently, there are three (T-SPOT.TB, QntiFERON-TB Gold and QFTGIT) commercial available
kits of IGRAs. T-SPOT.TB and QuantiFERON TB Gold
assays use Early Secretory Antigenic Target (ESAT)-6
and Culture Filtrate Protein (CFP)-10 as specific
antigens. Whereas, QFTGIT incorporates three Mycobacterium tuberculosis (Mtb) specific antigens (ESAT-6,
CFP-10 and TB7.7).
IGRAs have been widely evaluated in active TB
patients for the diagnosis of Mtb infection and various
levels of sensitivities and specificities have been reported
[9-16]. However, most of the information on the performance of tests has been reported from the developed
countries, where the prevalence of TB is moderate or
low. Thus, further information is needed on the performance of IGRAs from developing countries where TB is
endemic as well as other factors such as helminthic
infections, HIV/AIDS and undernutrition are prevalent
[17,18]. On top of these, IGRAs have been found to be
affected by factors like ethnic background and immune
status of the population being tested [19,20]. The importance of revising the current recommended cut-off
values of the tests based on evidence from various epidemiological and population context is also becoming
an important issue [21,22]. Therefore, it is of practical
importance to evaluate IGRAs in countries and population under different settings prior to its application for
routine screening of active TB or epidemiological study
of LTBI.
According to the World Health Organization (WHO)
estimate, Ethiopia is ranked 7th among the 22 countries
with high-burden of TB in the world, while it stands
second in Africa [3]. Nevertheless, to the best of our
Page 2 of 10
information, there is no published work that has evaluated the performance of QFTGIT for the diagnosis of
Mtb infection in Ethiopia. Thus, as part of a large study
on TB in Afar pastoral communities and their livestock,
we assessed the performance of QFTGIT in subjects
clinically suspected of PTB and apparently healthy individuals, using the cut-off value recommended by the
manufacturer and disease-specific cut-off value.
Methods
Study Sites and Subjects
The study was conducted at Dubti Referral Hospital
(DRH) and Awash Health Centre (AHC) in Afar region,
North-East Ethiopia between December 2008 and
March 2009. Dubti Hospital is a referral hospital for the
region and it is located in Lower Awash valley, approximately at 574 km to the North-East of Addis Ababa.
Awash Health centre is located in the Middle Awash
valley at 220 km to the East of Addis Ababa. The majorities (80%) of the Afar population are pastoralists and
they are distinct ethnic group [23]. TB is one of the
major public health problems in Afar and the region is
ranked 2nd in Ethiopia with a notification rate between
146-260 per 100, 000 population [3,24].
Patients who visited outpatient department (OPD) of
the two health facilities and who met the inclusion criteria were invited to participate in the study. Patients
were eligible if they were clinically suspected of active
PTB by physician, 18 years or above, volunteer to provide blood and sputum samples, volunteer to be tested
for HIV infection and volunteer to provide written
informed consent. Severely sick patients (include those
patients who could not able to walk, sit, speak or give
informed consent), anaemic patients and pregnant
women were excluded from the study. Patients with
known history of immunosuppressive therapies and who
were on anti-TB treatment were also excluded from the
study. Fifty five adult healthy pastoralists were also
recruited in the study as control group. The inclusion
criteria for the control group were: age between 18 and
30 years, TST negativity, absence of history of contact
with TB patients, absence of previous history of TB,
absence of symptoms of active TB, voluntariness to provide blood sample as well as to be tested for HIV infection and willingness to provide written informed
consent.
Socio-demographic data like gender, age, ethnicity and
occupation, as well as information on clinical/radiological findings and co-morbidities with other diseases were
recorded by physician during clinical investigation. All
study participants were also interviewed for previous
history of TB treatment, contact with TB patients and
BCG scar by physician. The study protocol was
approved by the Ethical Clearance Committee of the
Legesse et al. BMC Infectious Diseases 2010, 10:354
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Aklilu Lemma Institute of Pathobiology (ALIPB), Addis
Ababa University and the Regional Committee for Medical Research Ethics of Southern Norway. Upon recruitment, the aim of the study was described to the study
participants and written informed consent was obtained
from each of the study participant.
Sputum Collection and Examination
Three sputum specimens (spot-morning-spot), were
obtained from each study participant, who was clinically
suspected of PTB by a physician as previously recommended [25]. Briefly, the first sputum specimen was collected in the health facility (spot specimen) on the first
day the patient visited the health facility. The second
was an early morning sputum specimen (morning specimen), which was collected at home on the second day
and delivered on the same day to the laboratory. The
third sputum specimen was collected in the health facility (spot specimen) on the second day after delivering
the morning specimen. Smear was prepared from a portion of each specimen, processed by the Ziehl-Neelsen
(ZN) staining technique and microscopically examined
for acid-fast bacillus (AFB) as previously described [26].
The remaining specimen was stored at 4°C until transported to the ALIPB Microbiology laboratory under
cold condition. Upon reaching the laboratory, specimens
were pooled together, decontaminated for 15 minutes
with an equal volume of 4% NaOH and centrifuged at
3000 revolution per minute (rpm) for 15 minutes. The
supernatant was decanted while the sediment was neutralized with 1% (0.1N) HCl using phenol red as an indicator. Neutralization was achieved when the colour of
the solution was changed from purple to yellow. Then,
0.1 ml of the pellet was inoculated onto LowensteinJensen medium containing pyruvate or glycerol and
incubated for 10 weeks at 37°C [27]. Cultures were followed weekly for the growth of mycobaterial colonies
and positivity for AFB was confirmed by microscopy.
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indeterminate using QFTGIT analysis software developed by the company (QFTGIT, Cellestis Ltd., Carnegie,
Australia).
Determination of Specificity of the Test
The specificity of the test was assessed using blood sample collected from 55 apparently healthy adult pastoralists who were negative for LTBI by TST. They were also
seronegative for HIV infection.
Tuberculin Skin Testing in Healthy Control Subjects
Immediately following blood collection from the right
hand, 0.1 ml (2T.U/0.1 ml) Tuberculin PPD RT23 (Statens Serum Institute, Copenhagen, Denmark) was administrated intradermally in the middle third of the left
forearm by well experienced nurse. The diameter
induration transverse to the long axis of the forearm
was measured between 48-to-72 hours using flexible
plastic ruler. Diameter of skin induration < 6 mm was
considered as negative for the test as recommended by
the manufacturer.
Estimation of Disease-specific Cut-off Value
A receiver operator characteristic (ROC) curve analysis
was used to estimate disease-specific cut-off value for
QFTGIT using data of patients clinically suspected of
PTB and healthy subjects as previously described
[11,22].
Stool and Urine Samples Collection and Examination
Stool and urine samples were collected from the study
participants, processed by the Kato and filtration techniques, respectively. The stool samples were microscopically examined for ova of Schistosoma mansoni (Sm)/
other intestinal helminths, while urine samples were
microscopically examined for ova of Schistosoma haematobium (Sh) as previously described [28].
Data Analysis
QuantiFERON-TB Gold In-Tube Assay
The QFTGIT was performed according to the manufacturer’s instructions (QuantiFERON-TB Gold In-Tube,
Cellestis Ltd., Carnegie, Australia). Briefly, on the day of
sputum collection, 1 ml venous blood sample was collected from each individual directly into three tubes
(TB-specific antigens, mitogen and nil tubes). The samples were incubated for 24 hours at 37°C in the laboratory of each health facility and then centrifuged at
3000 × rcf (relative centrifugal force) for 10 minutes.
Plasma was collected and stored at 4°C until IFNg assay
was performed using ELISA. The optical density (OD)
of each test was read using a 450 nm filter with a
620 nm reference filter using ELISA plate reader, and
the results were interpreted as positive, negative or
Data were entered into EpiData Software version 3.1 and
analyzed using Stata version 8 (StataCorp LP). Means
were compared using the Student’s t test for two independent samples. Sensitivity and specificity of QFTGIT
were estimated using (a) the cut-off value recommended
by the manufacturer (≥0.35, IU/ml) and (b) ROC using
the data generated from the current study (disease-specific cut-off value). Sensitivity was calculated by dividing
the number of patients who were positive by culture
and QFTGIT by the number of true positives (i.e. culture positive). In the case of suspected patients of PTB,
the specificity of the test was evaluated by dividing the
number of individuals with a negative test both by the
QFTGIT and culture by the number of total negative (i.
e. culture negative). In the case of healthy subjects, the
Legesse et al. BMC Infectious Diseases 2010, 10:354
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specificity of the test was evaluated by dividing the
number of individuals with a negative test both by the
QFTGIT and TST by the number of total negatives by
the TST. The effect of socio-demographic variables on
the diagnostic performance of QFTGIT was evaluated
using univariable and multivariable logistic regression. P
value less than 0.05 was considered significant in all
analysis.
Results
Baseline Characteristics of Study Patients
A total of 167 (132 from DRH and 35 from AHC) study
participants who were clinically suspected of PTB were
requested to be involved in the study. Out of these subjects, 27(16.2%) did not volunteer to provide complete
data. Hence, data were analyzed for 140 subjects
(108 from DRH and 32 from AHC) whose age range
was between 18 and 70 years (mean age 34.2 years).
More than half of the study participants were males
(54.3%), belong to Afar ethnic group (72.9%) and pastoralists by occupation (56.4%).
Out of 140 subjects who were tested for HIV infection, 27 (19.3%) were seropositive. Fifty four (38.6%)
subjects had chest radiograph results of whom
28 (51.9%) had suggestive results for PTB. The rest,
86 (61.4%) subjects had no results on chest radiograph
as radiograph examination for these individuals was not
requested by the physician. Thirty four (24.3%) and
6 (4.3%) subjects reported previous history of contact
with TB patients and previous history of TB treatment,
respectively. Twenty eight (20.0%) subjects had BCG
scar.
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of the 37 culture confirmed PTB patients, 12 (32.4%)
were negative by QFTGIT. Out of these 12 subjects, 1
was seropositive for HIV infection. Among 28 participants who had positive chest radiography for PTB, 18
(64.3%), 6 (21.4%) and 4 (14.3%) had positive, negative
and indeterminate results by QFTGIT, respectively.
Results from logistic regression analysis showed that
individuals in the age group between 30 to 44 years (24
out of 47, 51.1%) were more likely to have a negative
QFTGIT results compared to those individuals who
were younger than 30 years (16 out of 60, 26.7%)
(Crude OR = 0.35; 95% CI, 0.16 to 0.78; p = 0.011,
adjusted OR = 0.39; 95% CI, 0.16 to 0.98; p = 0.046).
On the other hand, there was no significant association
between the results of QFTGIT and participants’ sociodemographic characteristics such as gender, ethnicity,
BCG scar or occupation (Table 1).
Sensitivity and Specificity of QFTGIT in HIV-Seronegative
study patients
Data from 27 participants who were seropositive for
HIV infection was excluded from the analysis of the
Table 1 Association of study participants’ sociodemographic characteristics with QFTGIT result
Characteristic
Gender:
Female (n = 64)
Male (n = 76)
Crude OR
(95%, CI)
Adjusted OR
(95%, CI)
Reference
Reference
1.30 (0.65-2.62)
1.15 (0.55-2.4)
Reference
Age (years):
18-29 (n = 60)
Reference
Bacteriological Assessment
30-44 (n = 47)
0.35 (0.16-0.78)
0.39 (0.16-0.98)
Out of the 140 study participants, 37 (26.4%) and
12 (8.6%) were positive for active PTB by culture and
direct AFB smear microscopy, respectively. Of the 12
study participants who had positive results by direct
AFB smear microscopy, 2 were negative by culture. Out
of the 37 subjects who had positive results by culture,
6 individuals were HIV-seropositive. Of the 28 individuals who had positive results by chest radiography,
4 (14.3%) were positive by culture.
45+ (n = 33)
0.84 (0.33-2.1)
0.79 (0.29-2.21)
Reference
1.81 (0.79-4.12)
Reference
0.80 (0.21-3.07)
Pastoralist (n = 79)
Reference
Reference
Other (n = 61)
1.63 (0.79-3.31)
1.49 (0.51-4.33)
Yes (n = 28)
Reference
Reference
No (n = 112)
2.37 (0.89-6.32)
1.45 (0.38-5.55)
Reference
Reference
2.9 (0.33-25.38)
1.49 (0.12-17.17)
Yes (n = 34)
Reference
Reference
No (n = 106)
1.75 (0.74-4.13)
1.71 (0.66-4.47)
Positive (n = 27)
Reference
Reference
Negative (n = 113)
0.77 (0.32-1.81)
0.75 (0.28-2.02)
IFNg Assay Using Cut-off Value Recommended by the
Manufacturer
Out of the 140 participants who were clinically suspected
of PTB, 83 (59.3%) were positive for Mtb infection by
QFTGIT using the cut-off value recommended by the
manufacturer. Seven (5%) individuals (6 from DRH and 1
from AHC) had indeterminate results. Out of the 27 participants who were seropositive for HIV infection, 15
(55.6%), 11(40.7%), and 1(3.7%) had positive, negative
and indeterminate results by QFTGIT, respectively. Out
Ethnicity
Afar (n = 102)
Other (n = 38)
Occupation :
BCG scar present
Previous TB treatment:
Yes (n = 6)
No (n = 134)
Contact with TB
patient:
HIV status:
Legesse et al. BMC Infectious Diseases 2010, 10:354
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sensitivity and specificity of QFTGIT with the assumption that infection with HIV could affect either the bacteriological or QFTGIT results. Similarly, data from 6
individuals who had indeterminate results was also
excluded from sensitivity/specificity analysis. Hence, the
performance of the test was assessed using data from
107 (31 culture positive and 76 culture negative) individuals who were clinically suspected of PTB. Out of the
107 subjects, 20 (18.7%) were positive both by culture
and QFTGIT, while 28 (26.2%) were negative by both
tests (Table 2). Using the manufacturer recommended
cut-off value, the sensitivity of the test was 64.5% (95%
CI, 45.4 to 80.8), while its specificity was 36.8% (95% CI,
26.1 to 48.7). The positive and negative predicative
values of the test were 29.4% (95% CI, 19.0 to 41.7) and
71.8% (95% CI, 55.1 to 85.0), respectively. Table 2
depicts the sensitivity and specificity of the QFTGIT at
various cut-off values of the level of IFNg.
When the disease-specific cut-off value was estimated
to be ≥ 0.2 IU/ml of IFNg level, the sensitivity of the
test increased from 64.5% to 67.7% (95% CI, 48.6 to
83.3), while the specificity was slightly decreased from
36.8% to 28.9% (95% CI, 19.1 to 40.5). Among 11
patients who were positive for PTB by culture, but negative by QFTGIT at the cut-off value ≥ 0.35 IU/ml, 1 was
found to be positive at the cut-off value ≥ 0.2 IU/ml.
Out of the 28 patients who were negatives by QFTGIT
at the cut-off value ≥ 0.35 IU/ml, 6 (21.4%) were found
to be positive using cut-off value ≥ 0.2 IU/ml. Among
32 healthy subjects who were negative by QFTGIT at
cut-off value ≥ 0.35 IU/ml, 10 (31.3%) were found to be
positive for LTBI using cut-off value ≥ 0.2 IU/ml of
IFNg level.
A cut-off value of ≥ 0.1 IU/ml of IFNg level increased
the sensitivity of the test from 64.5% to 77.4% (95% CI,
58.9 to 90.4), while the specificity decreased from 36.8%
to 23.7% (95% CI, 14.7 to 34.8). Of those 11 patients
who were positive for active PTB by culture but negative
by QFTGIT, 4 (36.4%) were found to be positive by
QFTGIT using cut-off value ≥ 0.1 IU/ml. Among
28 patients who were clinically suspected of PTB, but
negative both by culture and QFTGIT at the cut-off
value ≥ 0.35 IU/ml of IFNg level, 10 (35.7%) were found
to be positive using cut-off value ≥ 0.1 IU/ml. Similarly,
of the 32 healthy subjects who were negative at the cutoff value ≥ 0.35 IU/ml, 13 (40.6%) were found to be positive for LTBI using cut-off value ≥ 0.1 IU/ml IFNg level.
Sensitivity and Specificity of QFTGIT in HIV-seropositive
Study Patients
Out of the 27 HIV-seropositive subjects, 5 (18.5%) were
positive both by culture and QFTGIT, whereas 10
(37.0%) subjects were negative by both tests (Table 3).
All the 11 subjects who had negative results by
QFTGIT, had < 0.1 IU/ml of IFNg level. Hence, the sensitivity and specificity of QFTGIT were assessed at the
manufacturer recommended cut-off value. Data from
one subject who had indeterminate result was excluded
from the analysis. The sensitivity and the specificity of
the test were 83.3% and 50%, respectively.
Specificity of QFTGIT in Control Healthy Subjects
Out of 55 healthy adult pastoralists (14 females and 41
males, age range 18-30, mean age 23.6 years) who were
negative by TST, 23 (41.8%) were diagnosed as positive
for LTBI by QFTGIT. Hence, the specificity of QFTGIT
was found to be 58.2% (95% CI, 44.1 to 71.3).
Comparison of the Level of IFNg in HIV-seronegative
Study patients
Figure 1 shows the mean levels of IFNg induced by the
specific antigens (ESAT-6, CFP-10 and TB7.7) in three
sub groups (culture positive, culture negative and control healthy subjects) of the study participants. The
results of one-way analysis of variance showed that the
mean levels of IFNg induced by the specific antigens in
the three sub groups of the study participants were not
equal (F = 8.18; df = 2, p = 0.0004). Further pair-wise
analysis showed that the mean levels of IFNg was not significantly different in study participants with culture negative (n = 82) and culture positive (n = 31) (3.33 ± 4.43 IU/
ml vs 4.16 ± 4.72 IU/ml, p = 0.3850). Similarly, there was
Table 2 Diagnosis of Mtb infection by culture method and QFTGIT at various cut-off values of the level of INFg (IU/ml)
in HIV-seronegative study participants (n = 107)
Culture method
QFTGIT/cut-off value (IU/ml)
Diagnostic performance
Number of positive
Number of negative
Sensitivity (%)
Specificity (%)
Number of positive
20
48
64.5
36.8
Number of negative
11
28
≥0.2
Number of positive
21
54
67.7
28.9
≥0.1
Number of negative
Number of positive
10
24
22
58
77.4
23.7
Number of negative
7
18
≥0.35*
*Manufacturer’s recommended cut-off value.
Legesse et al. BMC Infectious Diseases 2010, 10:354
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Table 3 Diagnosis of Mtb infection by culture method and QFTGIT at cut-off value recommended by the manufacturer
in HIV-seropositive study participants (n = 26)
Culture method
QFTGIT/cut-off value (IU/ml)
≥0.35*
Diagnostic performance
Number of positive
Number of negative
Sensitivity(%)
Specificity(%)
Number of positive
5
10
83.3
50.0
Number of negative
1
10
*Manufacturer’s recommended cut-off value.
no statistically significant difference in the mean levels of
IFNg in participants who were positive both by culture
and QFTGIT (n = 20) and those individuals who were
negative by culture (n = 48), but diagnosed positive by
QFTGIT (6.41 ± 4.49 IU/ml vs 5.69 ± 4.48 IU/ml, p =
0.5473). On the other hand, the mean level of IFNg was
significantly higher in study participants (n = 113) who
were clinically suspected of PTB than that of the 55
healthy subjects (3.56 ± 4. 51 IU/ml vs 1.08 ± 1.90 IU/ml,
p = 0.0002) (Figure 1). The mean level of IFNg in those 68
study participants who were clinically suspected of PTB
and diagnosed positive by QFTGIT was significantly
higher than that of 23 healthy individuals who were
15
IFN-g IU/ml
13
11
9
7
5
3
1
positive for LTBI by QFTGIT (5.9 ± 4.46 IU/ml vs 2.46 ±
2.32 IU/ml, p = 0.0007). Similarly, a significant difference
was found between mean levels of IFNg of those study
participants who were negative by culture, but positive by
QFTGIT (n = 48) and those 23 healthy individuals who
were positive for LTBI by QFTGIT (5.69 ± 4.48 IU/ml vs
2.46 ± 2.32 IU/ml, p = 0.0018).
Figure 2 shows the mean levels of IFNg induced by the
mitogen in the three sub groups of the study participants.
The results of one-way analysis of variance showed that
the mean levels of IFNg induced by the mitogen in the
three sub groups of the study participants were not equal
(F = 20.51; df = 2, p < 0.001). Further pair-wise analysis
showed that the mean levels of IFNg induced by the
mitogen were not significantly different in study participants with culture negative (n = 82) and culture positive
(n = 31) (8.21 ± 4.44 IU/ml vs 8.63 ± 4.89 IU/ml, p =
0.6605). In contrast, the mean level of IFNg induced by
the mitogen was significantly higher in 55 control healthy
subjects than in 113 study participants who were clinically suspected of PTB (12.6 ± 2.80 IU/ml vs 8.33 ± 4. 55
IU/ml, p < 0.001). The mean level of IFNg induced by
the mitogen was significantly higher in 55 control healthy
subjects than that of 82 culture negative subjects (12.6 ±
2.80 IU/ml vs 8.21 ± 4.44 IU/ml, P < 0.001) and also
than in 31 culture positive subjects (12.6 ± 2.80 IU/ml vs
8.63 ± 4.89 IU/ml, p < 0.001).
-1
CN(n = 82)
HS (n = 55)
Figure 1 Level of IFNg induced by specific antigens in three
groups of study participants. Whole blood samples were
collected from study participants suspected of pulmonary
tuberculosis and apparently healthy subjects directly into tubes
containing TB-specific antigens, mitogen and nil. The samples were
incubated for 24 hours at 37°C, plasma was collected and IFNg assay
was performed using ELISA. The optical density (OD) of each test
was read using a 450 nm filter with a 620 nm reference filter using
ELISA plate reader, and the level of IFNg (IU/ml) was interpreted
using QuantiFERON-TB Gold In-Tube (QFTGIT) analysis software
developed by the company (Cellestis Ltd., Carnegie, Australia). The
subjects were categorized in to three groups based on sputum
culture results as CP = sputum culture positive, CN = sputum
culture negative and HS = healthy subjects and the mean levels of
IFNg IU/ml were compared for the three groups. Each dot
represents the concentration of IFNg IU/ml of each individual and
the solid line shows the mean level of the IFNg.
IFN-g IU/m l
CP (n = 31)
17
15
13
11
9
7
5
3
1
-1
CP(n=31)
CN(n=82)
HS(n=55)
Figure 2 Level of IFNg induced by mitogen in three groups of
study participants.
Legesse et al. BMC Infectious Diseases 2010, 10:354
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Stool and Urine Examination
Out the total 140 subjects who were clinically suspected
of PTB, 126 (90.0%) provided stool samples and 140
(100%) provided urine samples. In this group of study
participants, only 2 (1.4%) individuals were positive for
Sh infection. These 2 individuals were also positive by
QFTGIT. Out of the 55 healthy individuals, 9 (16.4%)
were positive for Sh infection, of whom 4 were positive
for LTBI by QFTGIT.
Discussion
In this study, we evaluated the diagnostic performance
of QFTGIT in subjects clinically suspected of PTB using
culture result as gold reference in area where the disease
is highly prevalent [3,24]. Using the manufacturer
recommended cut-off value; we found sensitivity of
64.5% and specificity of 36.8% of the test. The sensitivity
of the test observed in the present study is much lower
than sensitivity reported from TB endemic areas using
the same test [9,29]. The discrepancy between the present study and these previous studies could be explained
by multiple factors like population background and
immune status, the clinical (disease) status and the
socio-demographic characteristics of the study participants [14,19,20,30]. In the present study, 11 HIV-seronegative individuals who were positive for active PTB by
culture were found to be negative by QFTGIT at cut-off
value recommended by the manufacturer. Hence, the
false negativity rate of the test was 35.5%, which
is higher than the false negative rates reported
for QFTGIT or other IGRAs by previous studies
[9,14,20,31,32]. False negative results related to IGRAs
have been associated with various factors such as
impaired immune status, age of patients, the infecting
Mtb strains, genetic background of the population or
other chronic diseases [19,20,31-33]. Although QFTGIT
was performed according to the manufacturer’s instructions, factors like inadequate shaking of blood samples,
incubation delay and power outages during incubation
of blood samples are also inevitable assay-related problems which could be another possible explanation for
the false-negative results of the test [34]. Hence, further
studies are warranted to investigate factors contribute to
false negative results of the QFTGIT in the present
study area.
Study done on the evaluation of cut-off values of
IGRAs in the diagnosis of Mtb infection in Turkey
revealed that reducing the cut-off value to ≥ 0.1 IU/ml
of IFNg improved the sensitivity of the test by 13.8%
[22]. Lee and his co-workers [11] also showed an
improved sensitivity (from 70.1% to 86.2%) of the test
by reducing the cut-off value to ≥ 0.13 IU/ml of IFNg.
Study in Zambian adults [32] also showed an increased
sensitivity of the test from 74% to 82 by using a cut-off
Page 7 of 10
value ≥ 0.13 IU/ml of IFNg. Study in Brazilian TB
patients [32] also showed an increased sensitivity by
10% by lowering the cut-off value to 0.2 IU/ml of IFNg.
In the present study, reducing of the cut-off value to ≥
0.1 IU/ml of IFNg increased the sensitivity of QFTGIT
from 64.5% to 77.4%. Thus, it is also reasonable to suppose that false negative results could occur because of
the present high cut-off value of the test recommended
by the manufacturer.
Studies showed that lowering of the cut-off value
recommended by the manufacturer could decrease the
specificity of the test based on the epidemiological status
of the disease, though it improves sensitivity [11,22].
Some studies suggested the importance of reducing the
present cut-off point recommended by the manufacturer
for the diagnosis of active TB in areas where TB and
HIV are prevalent regardless of its specificity [21,22,31].
In our study, out of those 11 patients who were positive
for active PTB by culture, but negative by QFTGIT, 4
(36.4%) were found to be positive by QFTGIT using
cut-off value ≥ 0.1 IU/ml. Similarly, among 28 patients
who were clinically suspected of PTB, but negative by
culture and QFTGIT at the cut-off value ≥ 0.35 IU/ml
of IFNg level, 10 (35.7%) were found to be positive using
cut-off value ≥ 0.1 IU/ml. Logically, a test with high specificity is important in areas with a high prevalence of
TB in order to minimize unnecessary treatment with
anti-TB drugs. Nonetheless, taking into consideration
the burden of TB in the present study area, we maintain
the previous notion [21,22,31] that using low cut-off
value of QFTGIT could aid for the screening of active
TB in combination with clinical features though further
studies on the immune profile of the Afar pastoralists
are important.
Because of the lack of a gold standard test that accurately identifies subjects without LTBI, many investigators have evaluated only the sensitivity of the IGRAs
either in culture proven or using combined results of
culture and clinical findings as gold reference [14,20,35].
In the present study, we observed very low specificity of
QFTGIT in patients clinically suspected of PTB compared to the specificity reported from elsewhere [9].
This poor specificity could be due to the high prevalence of TB in the present study area [3].
We also found a low specificity of QFTGIT in TST
negative healthy subjects which is inconsistent with previous studies which showed high specificity rates of
IGRAs in TST negative healthy individuals [10,11,32].
Among 55 healthy subjects who had skin induration < 5
mm, 23 (41.8%) subjects were found positive for LTBI
by QFTGIT at the cut-off point recommended by the
manufacturer. This could be explained by several factors
such as: 1) False-negative results/low sensitivity of TST
in subjects truly infected with Mtb, but un-reactive due
Legesse et al. BMC Infectious Diseases 2010, 10:354
http://www.biomedcentral.com/1471-2334/10/354
to diminishing of the response over time [36]. 2) Undernutrition associated immunosuppression [37]. 3) The
difference in the potential of the antigens (ESAT-6,
CFP10) to stimulate specific IFNg secreting effector Tcells [38]. 4) Although TST was performed by experienced nurse, it is impossible to exclude the effect of
technical-related factors like errors in administration of
the tuberculin or incorrect reading of the skin induration on the performance and interpretation of the
results. Thus, further study is needed to elucidate factors
hampered the sensitivity of TST, as compared to high
sensitivity of the QFTGIT in the present study area.
In this study, we also observed a low positive predictive value of QFTGIT compared to reports from a low
endemic setting [39]. This low positive predictive value
of the test might be explained mainly by the high prevalence of LTBI in the present study area. Study showed
that IGRAs are more sensitive in the diagnosis of
patients clinically suspected of active TB than culture
method [40]. In the present study, among 68 subjects
who were positive by QFTGIT, 48 (70.6%) were found
negative by culture method. Since culture method may
not also absolutely classify all clinically suspected
patients as patients with and without active TB, it is reasonable to presume that culture method could result in
false negative which could affect both the sensitivity and
the true positive predictive value of the test.
In the present study, QFTGIT revealed an overall
positivity rate of 59.3% in patients clinically suspected of
PTB. This positivity rate had no association with
patients’ baseline characteristics such as gender, ethnicity, occupation or HIV status. Study showed that HIVinfection increases the risk of indeterminate and false
negative results by QFTGIT in active TB patients,
whereas it decreases the sensitivity of the test, especially
in patients with low CD4+ cell count [31]. In this study,
indeterminate/false negative results or insensitivity of
QFTGIT was not associated with HIV-seropositivity.
This finding indicates that QFTGIT could help in the
screening of LTBI as well as in the diagnosis of active
TB in HIV-infected subjects though evidence on the status of CD4+ cells is crucial [41].
In vitro analysis of the responses CD4 T cells to either
ESAT-6 or short-term culture filtrate (ST-CF) using
other immunological assays indicated that Mtb infection
induces high level of IFNg in asymptomatic healthy subjects with LTBI compared to that of patients with active
TB [30,42,43]. In this study, we found that the level of
IFNg induced by specific antigens in culture positive as
well as in clinically suspected patients was higher than
that of the control healthy subjects with LTBI. Previous
studies also demonstrated that T-cell responses to specific-mycobacterium antigens measured using IGRAs is
significantly higher in patients with active TB or
Page 8 of 10
clinically suspected cases than control healthy subjects
with LTBI [10,11,35]. These findings necessitate further
studies on the potential use of IGRAs for the diagnosis
of active TB in combination with bacteriological and
clinical features.
In this study, we could not involve a large number of
study participants because of the high cost of QFTGIT.
Hence, this small sample size of the study participants
might limit the generalization of the findings according to
the different subgroup of the study participants. Another
limitation of this study is the incapability to provide a reliable data on the specificity of the QFTGIT because of the
lack of gold standard for the diagnosis LTBI as well as the
TB high-burden setting of the study area. Nevertheless, we
believe that our study would provide important information on the performance of QFTGIT particularly in the
present study setting where data is not available, and generally in high TB endemic settings.
Conclusions
Our findings revealed a low sensitivity of QFTGIT in
the diagnosis of Mtb infection in the present study
population using the cut-off value recommended by the
manufacturer. Nevertheless, the sensitivity increased
from 64.5% to 77.4% by lowering the cut-off value
recommended by the manufacturer to ≥ 0.1 IU/ml of
IFNg. Hence, it is of practical importance to evaluate
the best disease-specific cut-off value that could improve
the sensitivity of QFTGIT in population under different
settings prior to its application for routine diagnosis of
active TB or epidemiological study of LTBI.
Acknowledgements
We are grateful to study participants, DRH and AHC health workers, Afar
Regional Health Bureau. We would like to thank staff of Armauer Hansen
Research Institute for their cooperation during laboratory work. The study
was financially supported by Norwegian Programme for Development,
Research and Education, NUFU (NUFU PRO-2007/10198).
Author details
1
Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis
Ababa, Ethiopia. 2Faculty of Veterinary Medicine, Addis Ababa University,
Bishofituu, Ethiopia. 3Department of General Practice and Community
Medicine, Institute for Health and Society, University of Oslo, Oslo, Norway.
Authors’ contributions
ML designed the study, participated in data collection, analysis and drafted
the manuscript. GA, participated in study design, data collection, analysis
and write-up. GM participated in study design, data collection and write-up.
GMD, participated in study design, data analysis/interpretation and write-up.
GB involved in study design and write-up manuscript. FA involved in study
design, data analysis and write-up of the manuscript and critically revised
the manuscript. All authors read and approved the final manuscript. ML is
the guarantor of the paper.
Competing interests
The authors declare that they have no competing interests.
Received: 8 April 2010 Accepted: 17 December 2010
Published: 17 December 2010
Legesse et al. BMC Infectious Diseases 2010, 10:354
http://www.biomedcentral.com/1471-2334/10/354
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/10/354/prepub
doi:10.1186/1471-2334-10-354
Cite this article as: Legesse et al.: Performance of QuantiFERON-TB Gold
In-Tube (QFTGIT) for the diagnosis of Mycobacterium tuberculosis (Mtb)
infection in Afar Pastoralists, Ethiopia. BMC Infectious Diseases 2010
10:354.
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